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Evolution: Rooting the Eukaryotic Tree of Life

The root of the eukaryotic tree is a major unresolved question in evolutionarybiology. A recent study marshals mitochondrial genes to place that rootbetween the enigmatic Excavates and all other eukaryotes, providing aninteresting new perspective on early eukaryotic evolution.

Tom A. Williams

Eukaryotes are the group of organismswhose cells contain a mitochondrionand a nucleus. Although we often thinkof eukaryotes as comprising animals,plants, fungi, and the single-celledprotists, molecular phylogenetics hasturned that view on its head [1]. At thelevel of the genome, eukaryoticbiodiversity is largely microbial, witheach of the familiar multicellular groupsarising independently from unicellularancestors [2]. In this emerging view,which is based mainly on phylogeneticanalyses of large sets of broadlydistributed genes [3–5], mosteukaryotes can be classified into one ofthree major lineages. These are theAmorphea (animals, fungi, andAmoebozoans such as the slime mouldDictyostelium), the Diaphoretickes(including plants and most otherphotosynthetic eukaryotes), and theExcavates — a diverse group ofunicellular eukaryotes that includesimportant parasites such as Giardia,Trichomonas, and Trypanosoma.Despite this rapid progress, some ofthe most interesting questions abouteukaryotic evolution remainunresolved. Foremost among theseis the position of the root of theeukaryotic tree, a critical pointabout which there is currently littleconsensus. A new study by He et al. [6]reported in this issue ofCurrent Biologyrepresents an important contribution tothis ongoing debate.

The root of a phylogenetic treeis fundamental to its biologicalinterpretation, defining the startingpoint and polarizing the subsequentdivergence events within the tree. Forthe eukaryotic tree, the root position iscritical for identifying the genes andtraits that may have been present in theancestral eukaryote, for tracing theevolution of these traits throughoutthe eukaryotic radiation, and forestablishing the deep relationshipsamong the major eukaryotic groups.

The root of the eukaryotes has beenstudied intensively in recent years,although as yet no consensus appearsin sight. Published analyses haveplaced the root within the Amorphea[7], between the Amorphea and allother eukaryotes [8], between theArchaeplastida (plants) and all othereukaryotes [9], or within the Excavates[10] — all with rather differentimplications for the history and theprocess of eukaryotic evolution.

One reason for this lack of agreementmay be the intrinsic difficulty of rootingthe eukaryotic tree. The greatevolutionary distance that separateseukaryotes from their closestprokaryotic relatives makes standardphylogenetic rooting approachesunreliable and prone to artefact. Thishas led authors to explore alternativeapproaches, such as using theinformation from gene duplications andlosses [7] or slowly evolving molecularcharacters such as gene fusions [9] toroot the tree. The problem is that whenthese alternative strategies disagree,it is difficult to determine the bestapproach. As an added difficulty, theinterior branches of the eukaryotic treeseparating the major groups oftenappear to be rather short, perhapssuggesting that the Amorphea,Diaphoretickes and Excavatesdiverged from each other over arelatively short period of time followingthe origin of eukaryotes — theso-called ‘big bang’ of eukaryoticevolution [11,12].

Despite these difficulties in inferringthe root, recent discoveries havefleshed out our understanding ofeukaryotic origins and the ancestraleukaryote. The balance of evidencenow favours a symbiogenic origin forthe eukaryotic cell, in which anAlpha-proteobacterium— the ancestorof the mitochondrion — formed apartnership with an Archaeon,whose trace in modern eukaryotesis found mainly in the geneticapparatus of DNA synthesis,

transcription and, particularly,translation [13]. This scenario impliesthat the mitochondrion was gainedbefore the radiation of all knowneukaryotes, suggesting thatmitochondrial genes might provide aninteresting source of information aboutthe eukaryotic root. The idea is that,since mitochondria are descendedfrom Bacteria, trees of mitochondrialgenes can be rooted on the bacterialbranch, polarizing the relationshipswithin the eukaryotic portion of thetree — an approach pioneered byDerelle and Lang [8] and developedin the recent study of He et al. [6].While this work is unlikely to settle thedebate for good, He et al. employstate-of-the-art methods, and theiranalyses focus attention on the mostimportant challenges remaining in thisfield.As a starting point for their analyses,

He et al. [6] screened genomes fromthe Amorphea, Diaphoretickes andExcavates for mitochondrial genes.They identified 37 genes which werefound in all, or almost all, eukaryotes,and whose closest prokaryotichomologues were among the Bacteria.They found that the protein productsof most of these genes were targetedto the mitochondria of moderneukaryotes, consistent with the ideathat the genes originally entered theeukaryotic lineage with the ancestor ofthe mitochondrion. Note that thesegenes are often not encoded by themitochondrial genome, because manymitochondrial genes have beentransferred to the nucleus duringeukaryotic evolution [14]. Importantly,He et al. were able to show that theirset of genes contained a consistentphylogenetic signal within theeukaryotes — that is, that theygenerally agreed on the structure of theeukaryotic tree. Since each gene on itsown did not provide much rootinginformation, the authors combined all37 genes and inferred a single tree.Assuming that the mitochondrion wasacquired before the diversification ofthe main eukaryotic lineages, this treecould then be rooted on the branchleading to the bacterial outgroups(Figure 1). Following this logic, He et al.

Ancestral eukaryote

Excavates

Diaphoretickes

Amorphea

Alpha-proteobacterium

mtDNA

Mitochondrion

mtDNA

Current Biology

Figure 1. Mitochondrial genes root the eukaryotic tree.

Genes that entered the eukaryotic lineage with the mitochondrial endosymbiont trace the sub-sequent evolutionary diversification of the eukaryotes. The recent study of He et al. [6] sug-gests that one of the deepest splits lies between the Excavates and all other eukaryotes.

Current Biology Vol 24 No 4R152

rooted the tree between the Excavatesand all other eukaryotes.

If correct, an Excavate root wouldreinforce the astonishing biodiversity ofmicrobial eukaryotes by placing all ofthe familiar multicellular groups —animals, plants, and fungi — on oneside of the eukaryotic tree. It would alsosupport an emerging view of the lastcommon eukaryotic ancestor as arelatively complex single-celledorganism that already possessed agene repertoire comparable in sizeand complexity to moderneukaryotes [15,16].

But with a number of incompatiblerootings now proposed for theeukaryotic tree, why should this newroot be preferred to any of thealternatives? One reason might be theapparent robustness of the rootingsignal from the 37 genes analyzed byHe et al. When the authors comparedsupport for the Excavate root with twoof the leading alternatives — a rooton the branch leading to Amorphea(the ‘unikont–bikont’ root) or toDiaphoretickes — they found that bothalternatives could be strongly rejected,even using only small subsamples oftheir combined alignment. Thissuggests a strong signal for theExcavate root, at least from the set ofmitochondrial genes considered. Butthe selection of those genes mayrepresent a potential caveat of thisstudy, or at least a subject for furtherwork. A recent study by Derelle andLang [8] applied a similar strategy to Heet al., usingmitochondrial genes to rootthe eukaryotic tree—but their analysesinstead supported a root on theamorphean branch. The difference

comes down to the definition ofmitochondrial genes: while He et al.emphasised bacterial genes thatcontain a congruent phylogeneticsignal and are broadly conservedacross eukaryotes, Derelle and Langscreened for eukaryotic genes ofalpha-proteobacterial affinity, basedon the idea that the mitochondrionis descended from an Alpha-proteobacterium. As a result of thesedifferent criteria, there was onlymoderate overlap between the genesused in the two studies. In particular,not all of the genes analyzed byHe et al.are alpha-proteobacterial, with sometracing their ancestry to other bacterialgroups. This does not necessarily ruleout a mitochondrial origin, because theancestral mitochondrial genome likelycontained genes horizontally acquiredfrom other prokaryotic groups, asis observed for the genomes ofcontemporary Alpha-proteobacteria[17]; in principle, genes obtained fromother Bacteria could also be used toroot the tree if acquired before theradiation of eukaryotes. He et al.argue that their approach allowedthem to better detect and removemitochondrial genes that hadundergone horizontal transfer duringeukaryotic evolution, which mightinterfere with root inference if includedin the analysis.

This is an exciting time for researchinto eukaryotic origins. The new studyof He et al. is a thorough and interestingcontribution to the rooting question,although it is unlikely to provide thefinal word on this topic. The fact thatsimilar approaches can produce verydifferent results indicates that we still

have much to learn about the earliestperiod of eukaryotic evolution, and willsurely stimulate further debate.

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Institute for Cell and Molecular Biosciences,University of Newcastle, Newcastle uponTyne NE2 4HH, UK.E-mail: tom.williams2@ncl.ac.uk

http://dx.doi.org/10.1016/j.cub.2014.01.026